1. Field of the Invention
The invention relates to protein separation and, more particularly, relates to an apparatus and process for the large scale separation and retrieval of proteins from a crude mixture of cellular matter.
2. Discussion of the Related Art
It is desirable for a wide variety of diagnostic and research procedures to separate and retrieve proteins from a cellular extract. The most popular technique heretofore used for such separation is known as continuous vertical gel electrophoresis or simply gel electrophoresis. Gel electrophoresis involves the separation and retrieval of enzymes or other proteins from complex mixtures by means of a differential migration of mixture components through a continuous gel medium under the imposition of an electric current. Examples of devices for performing gel electrophoresis are disclosed in U.S. Pat. No. 3,384,564 to Ornstein et al.; U.S. Pat. No. 3,579,433 to Dahlgren; and U.S. Pat. No. 4,877,510 to Chen. Each of these patents discloses a device employing lower and upper reservoirs defining lower and upper buffer chambers containing an ionic buffer solution and a conductive gel, respectively. The gel physically separates the two reservoirs. The upper buffer chamber of the device can be separated from the lower buffer chamber by at least one semipermeable membrane which permits the passage of ions but which prevents the passage of enzymes or other proteins being separated. When an electric current is conducted through such a device via positive and negative electrodes in the lower and upper buffer chambers, proteins migrate downwardly through the gel in the upper buffer chamber at different rates which are dependent upon the conductive properties of the individual proteins, thus separating into strata each containing one protein or one group of similar proteins. The thus stratified proteins continue to move downwardly through the gel in the upper buffer chamber until further movement is blocked by the semipermeable membrane, if present. The layers of proteins are then removed one by one, e.g., through pumping, before the next layer reaches the membrane.
Gel electrophoretic devices of the type described above typically can separate and retrieve proteins only on a very small scale and then at only a very slow pace. In fact, the typical device is capable of purifying only a few milligrams of proteins in several days or even weeks when used in conjunction with conventional partial purification procedures. This significantly hampers research or other procedures which require the retrieval of large volumes of proteins relatively quickly.
Proteins could not heretofore be retrieved at higher rates without encountering substantial difficulties. For instance, as the size of the electrophoretic apparatus is increased to accommodate greater volumes of cellular extracts, the surface area of the gel in the upper buffer chamber does not increase enough to permit sufficient heat transfer to dissipate satisfactorily the heat which is necessarily generated by the electrophoretic process. Since enzymes and other proteins are heat sensitive, the rate of electrophoresis must accordingly be maintained at or below a level above which the temperature of the enzymes or other proteins being retrieved may become denatured or otherwise inviable.
This problem can be alleviated to some extent by providing a cooling mechanism for reducing the temperature of the gel in the upper buffer chamber, but devices incorporating such cooling mechanisms are expensive and complex to manufacture and operate. In any event, even if an electrophoretic device were to be dimensioned to enhance heat transfer and/or cooled by a cooling device to facilitate larger scale electrophoresis without overheating the protein containing extract, the purification capacity of such a device would be inherently limited because the purification capacity of the gel is limited. This inherent clarification limitation necessarily delays the purification of proteins from the very crude extracts which serve as the base materials in typical electrophoretic processes.
Electrophoretic devices of the type described above also are operationally limited by the location of their negative electrodes. More specifically, such electrodes are typically immovably mounted within the upper buffer chamber or the associated funnel and thus require that the upper buffer chamber always be filled to a certain minimum level for operation.